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Id1基因在BMP-2维持椎间盘细胞软骨特性中的作用

, PP. 2011-2016

Keywords: 分化抑制因子,骨形态发生蛋白,髓核细胞

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Abstract:

目的构建Id1基因过表达和RNAi慢病毒载体,并以兔椎间盘髓核细胞为研究对象,观察Id1基因表达水平调整后,BMP-2诱导髓核细胞分泌软骨特性因子的变化。方法①设计针对Id1基因过表达和RNA干扰的序列,应用基因重组技术将目的基因片段连接到慢病毒载体,转染293T细胞后测定病毒滴度。②将2种慢病毒分别感染髓核细胞,QRT-PCR和Westernblot检测髓核细胞Id1mRNA和蛋白表达。③重组腺病毒AdBMP2感染髓核细胞,利用已制备的2种慢病毒再次感染细胞,QRT-PCR检测细胞表达软骨特性分子的能力。结果①慢病毒感染髓核细胞后,Id1基因的mRNA与蛋白的表达量与未感染病毒组(0.428±0.079)及空载病毒组(0.400±0.045)相比,过表达组(0.848±0.154)增加,RNAi组(0.115±0.014)下降,差异具有统计学意义(P<0.01),符合预期结果。②细胞内Id1的表达增加可促进colⅡ(0.407±0.083)和ACAN(0.284±0.074)的mRNA表达增加,与GFP组比较,差异具有统计学意义(P<0.01)。③ADBMP2处理细胞后,Id1的表达增加可使colⅡ(0.590±0.072)和ACAN(0.351±0.066)的mRNA表达进一步显著升高;Id1表达降低时,以上因子的表达被明显抑制[colⅡ:(0.244±0.060),ACAN:(0.168±0.034),差异具有统计学意义(P<0.01)]。结论Id1能够促进椎间盘细胞软骨特性分子的表达,细胞内Id1基因水平变化能够显著影响BMP-2诱导细胞分泌软骨特性因子,推测Id1是BMP-2维持椎间盘细胞软骨特性作用的关键因子。

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